This research program contains several related objectives designed to give information which will assist in our continuing quest to understand the role pteridine or folate coenzymes and substrates play in enzymatic systems. Structural factors commonly present in these coenzymes include a fully-reduced pyrazine ring and a substituent at the 5-position. During the course of an enzymatic reaction oxidation of the pteridine usually occurs and, in certain cases, the substituent is removed. Enzymatic systems, using such cofactors, currently under investigation in this laboratory include, dihydrofolate reductase, thymidylate synthetase, pteridine reductase with the associated phenylalanine or tyrosine hydroxylases and methionine synthetase. Novel procedures using affinity chromatographic techniques are being used to rapidly isolate these enzymes from crude bacterial and mammalian sources, thus enabling specific structural characterization to be achieved with the aid of active-site directed substrate analogs containing fluorescent, mercurated or photoaffinity labels. Experience gained from coupling folate compounds to high molecular weight polysaccharides for the preparation of the affinity column materials is being employed in the development of a class of antagonists containing Methotrexate bound to various inert polymeric supports which may have novel therapeutic benefits for neoplastic disorders. In addition a new route to convert the 4-hydroxylated pteridines into those containing a 4-amino substituent is under investigation.